Abstract.InnovativeapproachesareneededtocreatetherapeuticsthattargetHIV.Existingdrugscanprolong patientlifespanbytargetingmultiplefacetsofthevirallifecycle,butnext-generationtherapiesareneededthat actonnewtargets?especiallythosethatresistmutation?toimprovelong-termtherapeuticcomplianceand outcome.HIV-1TARRNAisavalidateddrugtargetthatresistsmutationstointeractwiththeviralproteinTat, thus giving rise to an RNA-protein complex essential for proviral transcription and HIV-1 propagation. So far, TAR has evaded discovery of compounds with sufficient affinity and selectivity to warrant pharmaceutical development. To address this challenge, we undertook a ?semi-design and protein evolution? approach that yielded many novel, high-affinity (KDs ~ 1.3 to 0.5 nM) TAR Binding Proteins (TBPs) using yeast display maturation.Wethendeterminedthe1.80Šresolutionco-crystalstructureofonevariant,TBP6.7,incomplex withTAR,revealingthatthemajorbindinginterfaceconsistsofevolvedloop?2-?3,whichreadsouttheTAR RNAmajorgroove.WehypothesizethatcyclicpeptidescomprisingtheTBP6.7?2-?3loop,orotherTBPloops evolved in our lab, will be entry points to create a novel class of TAR binders. Indeed, the TBP6.7 ?2-?3 hairpinretainsaffinityandspecificityforTARwhenfusedtothesmallproteinSUMO,signifyingthatthe?2-?3 loopisnecessaryandsufficientforTARrecognition.Structuralidentificationofthe?-hairpinmotif,andouruse ofsemi-designandevolutionmakeourapproachfundamentallydifferentfromprioreffortstoblocktheTat-TAR interaction, while providing a robust experimental premise to pursue our aims: (Aim 1) Validate the observed TBP6.7-TAR interface and determine additional novel co-crystal structures of other TBPs evolved in our lab;? (Aim2)synthesizeandoptimizecyclicpeptidesderivedfromAim1thatbindTARandinhibititsinteractionwith Tat;?(Aim3)TestcyclicpeptidesfromAim2usingviralinfectivityassaystoinvestigatemechanismsofaction, therapeutic indices, and pharmacological properties in animals. To our knowledge, no other group has used protein evolution and structural biology to develop HIV-1 TAR-targeted reagents. We are a team of experts, comprising two P.I.s, with strong records in protein evolution, peptide-based drug discovery, HIV therapeutic discovery, measuring cell penetration and toxicity of biologics (McNaughton), and structural biology of therapeutically-relevant RNAs, protein-RNA complexes, and biophysical analysis of protein-RNA interactions (Wedekind), as well as two collaborators: Harold Smith (University of Rochester), a leader in drug discovery anddevelopment,andCEOofOyaGenInc.,aprivatecompanydevelopinganti-HIVdrugs,andDanGustafson (Colorado State University), a clinician and pharmacologist with expertise in measuring pharmacological profilesoftherapeutics.Weareuniquelyqualifiedandwellsuitedtoperformthiswork.High-valueoutcomes include: (i) identification of novel lead inhibitors of HIV, and (ii) validation of our ?semi-design? and structural approach,whichhasthepotentialforsustainedimpactonthedrugdiscoveryandinhibitordesignfields.